Transformation of 8-prenylnaringenin by Absidia coerulea and Beauveria bassiana

Article abstract of DOI:10.1016/j.bmcl.2012.08.060

Beauveria bassiana AM278 and Absidia coerulea AM93 converted 8-prenylnaringenin (1) into two glycoside derivatives (7-O-β-d- glucopyranoside) (2) and 7-O-β-d-4?-O-methylglucopyranoside) (3) in high yields in processes conducted in Saboraud medium. 8-Prenylnaringenin 7-O-β-d-4?-O-methylglucopyranoside (3) is a new compound. 8-Prenylnaringenin-7-sulfate (4) was obtained in transformation of 1 by Absidia coerulea AM93 in a buffer. Formation of conjugated products in this study proceeds in a manner analogous to mammalian systems which indicates the potential use of microbes to mimic mammalian metabolism.

Full text of DOI:10.1016/j.bmcl.2012.08.060

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6451–6453
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Transformation of 8-prenylnaringenin by Absidia coerulea
and Beauveria bassiana
⇑
Agnieszka Bartma n´ ska , Tomasz Tronina, Ewa Huszcza
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Beauveria bassiana AM278 and Absidia coerulea AM93 converted 8-prenylnaringenin (1) into two glyco-
Received 19 April 2012
Revised 13 August 2012
Accepted 14 August 2012
Available online 24 August 2012
000
side derivatives (7-O-b-
yields in processes conducted in Saboraud medium. 8-Prenylnaringenin 7-O-b-
D
-glucopyranoside) (2) and 7-O-b-
D
-4 -O-methylglucopyranoside) (3) in high
000
D
-4 -O-methylglucopyr-
anoside (3) is a new compound. 8-Prenylnaringenin-7-sulfate (4) was obtained in transformation of 1
by Absidia coerulea AM93 in a buffer. Formation of conjugated products in this study proceeds in a man-
ner analogous to mammalian systems which indicates the potential use of microbes to mimic mamma-
lian metabolism.
Keywords:
8
-Prenylnaringenin
Ó 2012 Elsevier Ltd. All rights reserved.
Glycosidation
Sulfation
Absidia coerulea
Beauveria bassiana
8
-Prenylnaringenin (1) is the most potent phytoestrogen iso-
In our previous study 30 fungi cultures were screened for po-
1
lated to date. Some authors suggest that the flavonoid may have
beneficial effects in postmenopausal women and may become an
alternative to classical hormone replacement therapy (HRT) treat-
tential 8-prenylnaringenin (1) transformation activity and several
9
microorganisms proved capable of such a reaction.
Absidia coerulea AM93 and Beauveria bassiana AM27810 were se-
lected for scale-up studies, because they gave the products with
the highest efficiency compared to the other tested fungi. This
study focused on identification of the metabolites of these
processes.
2
ment regimes. Although the molecular mechanism of action of
phytoestrogens is not yet fully understood, the use of phytoestro-
genic nutraceuticals, driven by society’s preference for what it con-
siders to be a more ‘natural’ and hence safer alternative to HRT, has
gained popularity.
Cultivation of fungi, isolation of xanthohumol from spent hops,
chemical cyclization of xanthohumol into isoxanthohumol,
demethylation to 8-prenylnaringenin (1), reaction work-up, and
product analysis were performed as described by Bartma n´ ska
Compound 1 Naturally occurs in hops3 and different Wyethia
4
species, but its content is too low to be of concern upon ingestion.
Phytoestrogens are normally taken up with food, for example 8-
prenylnaringenin (1) is present in beer up to 240
5
11
l
g/L, absorbed
et al. The experiments for biotransformation of 1 were conducted
in the gastrointestinal tract, and excreted in the urine. Most human
metabolites of 1 were produced by oxidation of two terminal
methyl groups, and the double bond on the prenyl group. Among
the identified metabolites there were also glucuronide and sulfate
in a Sabouraud medium. Additionally, we performed the experi-
ments of biotransformation of 1 in a phosphate buffer.12 Com-
pound 1 was obtained by demethylation of isoxanthohumol as
reported by Anioł et al. and its authenticity was confirmed by
NMR data.
1
3
6
,7
conjugates of 1.
Although prenylflavonoids have received much attention due to
their health-promoting properties, little is it know about the
metabolism of this class of molecules. The main goal of the present
work was to obtain new metabolites of 8-prenylnaringenin (1) by
transformation in fungi cultures. Our studies using microbes can
also be useful to mimic and predict mammalian metabolism of this
xenobiotic compound (1).8
Incubation of A. coerulea AM93 with 1 (70 mg) for 5 days (pH of
the culture 4.07) led to metabolite 2 in 49.3% yield (52.6 mg). The
product was isolated as dark orange crystals.
1
4
A UV spectrum of 2 showed maximum absorption bands at 284
and 345 nm, similarly to that of 1 (290 and 335 nm, respectively),
which indicated that the new compound was also a flavanone
1
13
derivative. In the H NMR and C NMR spectra of 2 some new
signals appeared, compared to 1. Six characteristic carbon signals
corresponding to a sugar moiety were observed in the region from
⇑
Corresponding author. Tel.: +48 713205197; fax: +48 713207744.
E-mail address: agnieszka.bartmanska@up.wroc.pl (A. Bartma n´ ska).
d
C
63.525 to 102.47 ppm, along with proton signals of d
H
ranging
1
from 3.47 to 5.075 ppm. Additionally, in the H NMR spectrum of
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.060

6
452
A. Bartma n´ ska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6451–6453
product 2 there is no C7-OH signal, whereas the H-6 signal is
shifted downfield by 0.26 ppm (from d 6.04 to d 6.296). The HMBC
To our knowledge, microbial 7-sulfation of 8-prenylnaringenin
(1) has never been reported before.
Sulfates of many natural flavones show antioxidant activity
and inhibit lens aldose reductase. Nikolic et al. studied the
000
25
correlation (H-1 /C-7) suggested the location of the sugar group at
0
00
26
7
C-7, and the coupling constants of the anomeric proton H-1
d = 5.075, J = 7.3 Hz) indicated a b-O-glycosidic linkage. An HR-
(
metabolism of 1 using Caco-2 cell monolayers and human hepato-
+
0
ESI-MS analysis of compound 2 showed the [M+H] peak at m/z
03.1932, which is in agreement with the formula of C26
On the basis of the above analysis, metabolite 2 was identified as
-prenylnaringenin 7-O-b- -glucopyranoside.
The spectroscopic data of 2 are in agreement with those re-
ported for Rhizopus oryzae KCTC6399 8-prenylnaringenin metabo-
cytes as model systems. In human hepatocytes 7-O- and 4 -O-glu-
0
5
30
H O
10
.
curonide conjugates were detected, but not 7- and 4 -sulfates,
which contrast with the Caco-2 cells that formed abundant sulfate
metabolites.
8
D
B. bassiana transformed 1 into the same product 3 in both the
buffer and the Saboraud medium, but the time of the reaction in
the buffer was longer. From the 12-day transformation extract
(pH 6.74) we isolated metabolite 3 in 34.0% yield.
1
5
lite. This flavonoid naturally occurs in two species of plants: (S)
fellodensin-F in Phellodendron japonicum and (R) fellodensin-F in
Phellodendron chinese var. Glabriusculum.16
27
B. bassiana is a highly regioselective bioreagent. We did notice
Transformation of 8-prenylnaringenin (1) (70 mg) by Beauveria
bassiana AM278 was carried out for 6 days (pH of the culture 3.51)
to give after purification 36 mg of product 317 as light yellow crys-
tals (yield 32.9%).
this in our previous research where we observed glycosylation of
2
8
11
29
naringenin, isoxanthohumol and xanthohumol in analogous
30
position. It was also described in the literature by Kim and Lee
and Herath et al.
3
1
The 1H and C NMR spectra of metabolite 3 are similar to 2,
13
In conclusion, we isolated two metabolites of 8-prenylnaringe-
nin (1) produced by A. coerulea (2 and 4) and one produced by B.
bassiana (3). We observed O-glucosylation in Sabouraud medium
and sulfate conjugation in a phosphate buffer. Microbial 7-sulfa-
tion of 1 has never been reported before. B. bassiana transformed
000
with additional signals indicating a 4 -O-methylglucopyranoside
0
00
unit [d
H
= 5.059/5.034 (1H, dd, J = 15.2, 7.7 Hz., H-1 ) and
000
13
d
H
= 3.563 (3H, s, 4 -OCH )]. In C NMR, apart from the reso-
3
nances of 1 structure, the presence of the O-methylated glycoside
moiety is confirmed by the following signals: an anomeric carbon
0
00
the substrate (1) to 7-O-b-
D-4 -O-methylglucopyranoside deriva-
atom (d
= 75.9, 78.999), a methoxymethine one (d
an oxygenated methine one (d = 78.143), a hydroxymethylene
one (d = 62.967), and an O-methyl carbon (d = 61.533). Com-
pound 3 showed a [M+H] peak at m/z 517.2097 in HR-ESI-MS,
which is in agreement with formula C27
These spectral data allow identifying metabolite 3 as 8-pren-
C
= 102.215/102.101), two hydroxymethine carbons
tive (3) in both of the media.
(d
C
C
= 80.808/80.787),
Conjugation reactions represent one of the major mechanisms
of detoxification of phenolics in animal tissues, named phase II
metabolism. Demonstration of parallelism between mammalian
and microbial systems is of considerable importance for investiga-
tion of drug metabolism. Glycosylation and sulfation enhances
hydrophilicity of flavonoids, which decreases their intracellular
compartmentation and their antimicrobial activity.
C
C
C
+
32 10
H O .
000
ylnaringenin 7-O-b-D-4 -O-methylglucopyranoside. To the best of
our knowledge, this compound has never been reported before.
In order to determined if the glycosylated products can be formed
in a medium which does not contain glucose we also performed the
experiments of biotransformation of 1 in a phosphate buffer.
Transformation of 1 by Absidia coerulea AM93 was carried out
for 9 days (pH of the culture 6.29–6.41) to give after purification
Our studies demonstrate the use of microbial transformation to
prepare the new metabolite (3) from 1.
Acknowledgments
This research was co-financed by the European Union from the
European Regional Development Fund under Operational
Programme Innovative Economy. Biotransformation for Pharma-
ceutical and Cosmetic Industry POIG.01.03.01-00-158/09.
1.8 mg of product 41 as yellow solid (31.1% yield). In the
H
8
1
2
NMR spectrum of 4 an absence of the signal of 7-hydroxyl proton
suggested conjugation reaction, but no additional signals were ob-
served (no glycosylation). The rest of signals were similar to 1, ex-
cept for H-6 signal, which was shifted downfield by 0.86 ppm.
1
3
References and notes
Comparison of C NMR data of the metabolite 4 and substrate
1) showed that while the C-7 signal was shifted upfield by
(
1.
Milligan, S. R.; Kalita, J. C.; Heyerick, A.; Rong, H.; De Cooman, L.; De Keukeleire,
3
3
.52 ppm, the signals of C-8 and C-6 were shifted downfield by
D. J. Clin. Endocrinol. Metab. 1999, 84, 2249.
2. Rad, M.; Humpel, M.; Schaefer, O.; Schoemaker, R. C.; Schleuning, W. D.; Cohen,
.81 and 5.03 ppm, respectively. Compound 4 showed a pseudo-
+
A. F.; Burggraaf, J. Br. J. Clin. Pharmacol. 2006, 62, 288.
molecular ion peak at m/z 465.0606 [M+2Na] (calcd for
3
4
.
.
Stevens, J. F.; Ivanic, M.; Hsu, V.; Deinzer, M. L. Phytochemistry 1997, 44, 1575.
McCormick, S.; Robson, K.; Bohm, B. Phytochemistry 1986, 25, 1723.
C
20
H
19
O
8
Na
2
S, 465.0591) in the positive-ion mode HR-ESI-MS, cor-
S. To confirm the presence of a
responding to the formula C20
H
19
O
8
5. Tekel, J.; De Keukeleiere, D.; Rong, H. J.; Daeseleire, E.; Van Petegem, C. J. Agric.
Food Chem. 1999, 47, 5059.
sulfate group, the product was hydrolyzed by treatment with acid
to yield the aglycone (1) [19, modified].
6.
Nikolic, D.; Yongmei, L.; Chadwick, L. R.; Grubjesic, S.; Schwab, P.; Metz, P.; van
Breemen, R. B. Drug Metab. Dispos. 2004, 32, 272.
Therefore, the structure of the more polar compound was iden-
tified as 8-prenylnaringenin-7-sulfate (4).
The test performed in the buffer suggested that the glucopyran-
osyl moiety in the metabolite 2 originated from the medium. In the
absence of glucose in the culture of A. coerulea the sulfation of sub-
strate (1) occurs.
7. Nikolic, D.; Yongmei, L.; Chadwick, L. R.; van Breemen, R. B. Pharm. Res. 2006,
3(5), 864.
2
8
9
.
.
Smith, R. V.; Rosazza, J. P. Arch. Biochem. Biophys. 1974, 161, 551.
Bartma n´ ska, A.; Tronina, T.; Huszcza, E. Z. Naturforsch. 2010, 65c, 603.
10. The microorganisms used in this study (Absidia coerulea AM93 and Beauveria
bassiana AM278) were purchased from Institute of Biology and Botany of the
Wrocław Medical University.
1
1. Bartma n´ ska, A.; Huszcza, E.; Tronina, T. J. Mol. Catal. B: Enzym. 2009, 61,
Conjugation with sulfate represents one of the major
mechanisms of detoxification of phenolics in animal tissues.
However, this reaction in microbial systems is rare and research
reports on this topic are limited. Some of them include: sulfation
221.
12. The cultures were shaken at 25 °C in 300-ml Erlenmeyer flasks, each
containing 100 ml of the Sabouraud medium. The biomasses of A. coerulea
AM93 5-day culture and B. bassiana AM278 3-day culture were centrifuged,
3
washed with the sterile phosphate buffer (0.1 mol/dm , pH 5.4) and suspended
20
21
of phenolic hydrocarbons, naringenin and 7-O-methylnaringe-
in 100 ml of the same sterile buffer. A solution of 8-prenylnaringenin (1)
(70 mg dissolved in 8 ml methanol) was equally distributed into four flasks
containing the tested fungus (17.5 mg of 1 per flask). The procedure was
identical for both tested strains. The next steps were the same as described
2
2
nin by Cunninghamella elegans, 5-hydroxyflavone by Streptomyces
2
3
fulvissimus, and cannflavin A and B by Mucor ramannianus and
Beauveria bassiana respectively.24
11
before.

A. Bartma n´ ska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6451–6453
6453
_
1
1
3. Anioł, M.; Szyma n´ ska, K.; Zołnierczyk, A. Tetrahedron 2008, 64, 9544.
4. 8-Prenylnaringenin 7-O-b- -glucopyranoside (2) was obtained as dark orange
crystals; (52.6 mg, 49.3%). UV kmax (MeOH) nm: 284, 345; H NMR (acetone-
18. 8-Prenylnaringenin-7-sulfate (4) was a yellow solid (21.8 mg, 31.1%). UV kmax
1
D
(MeOH) nm: 281, 346 (sh); H NMR (acetone-d
6
, 600 MHz), d: 11.90 (1H, s, 5-
1
0
0
0
OH), 8.56 (1H, s, 4 -OH), 7.41 (2H, d, J = 8.6 Hz, H-2 ,6 ), 6.96 (1H, s, H-6), 6.90
0
0
0 0
d
6
6
): d = 12.092/12.078 (1H, s, 5-OH), 7.409/7.405 (2H, d, J = 8.5 Hz, H-2 ,6 ),
.905/6.903 (2H, d, J = 7.4 Hz, H-3 ,5 ), 6.296 (1H, s, H-6), 5.494/5.473 (1H, t,
J = 2.9 Hz, H-2), 5.210 (1H, t, J = 7.4 Hz, H-2), 5.075 (1H, dd, J = 15.3, 7.2 Hz, H-
), 3.899 (1H, dd, J = 11.8, 2.2 Hz, H-6 ) 3.723 (1H, dd, J = 11.9, 5.5 Hz, H-6 ),
.605 (1H, m, H-5 ), 3.550 (1H, m, 2 ), 3.545 (1H, m, 3 ), 3.470 (1H, m, H-4 ),
.208 (1H, m, 1 ), 3.184 (1H, m, H-3ax), 2.828/2.800 (1H, t, J = 3.0 Hz, 3 equiv),
.606 (3H, s, 5 ), 1.606 (3H, s, 4 ). C NMR (acetone-d
65.361/165.332 (C-7), 163.970/163.987 (C-5), 161.337 (C-9), 159.626 (C-4 ),
(2H, d, J = 8.6 Hz, H-3 ,5 ), 5.45 (1H, dd, J = 12.8, 2.9 Hz, H-2), 5.15 (1H, t,
0
0
00 00
J = 7.3 Hz, H-2 ), 3.24 (2H, d, J = 6.8 Hz, H-1 ), 3.17 (1H, dd, J = 17.1, 12.9 Hz, H-
0
0
3ax), 2.77 (1H, dd, J = 17.0, 3.0 Hz, H-3 equiv), 1.58 (3H, s, H-4 ), and 1.58 (3H,
0
00
000
000
000
00 13
1
3
3
1
1
1
3
9
s, H-5 ). C NMR (acetone-d , 600 MHz), d: 199.43 (C-4), 162.82 (C-7), 162.30
6
000
000
000
0 00 0 0 0
(C-5), 161.29 (C-9), 159.61 (C-4 ), 132.00 (C-3 ), 129.84 (C-1 ), 129.84 (C-2 ,6 ),
0
0
00 0 0
124.87 (C-2 ), 117.12 (C-3 ,5 ), 113.14 (C-8), 105.90 (C-10), 102.43 (C-6), 80.71
+
0
0
00
13
00
00
00
6
): d = 199.273 (C-4),
(C-2), 44.69 (C-3), 26.94 (C-5 ), 23.87 (C-1 ), 19.00 (C-4 ). HR-ESI-MS [M+H] :
(m/z) 465.0606 (calcd for C20 Na S + 2Na: 465.0591.
0
H
19
O
8
2
00
0
0
0
00
32.217 (C-3 ), 131.870 (C-1 ), 129.875 (C-2 ,6 ), 124.789 (C-2 ), 117.150 (C-
,5 ), 111.306/111.255 (C-8), 105.434/105.399 (C-10), 102.47/102.36 (C-1 ),
7.247 (C-6), 80.796 (C-2), 79.020 (C-5 ), 78.954 (C-3 ), 75.651 (C-2 ), 72.212
19. Mann, P.; Tofern, B.; Kaloga, M.; Eich, E. Phytochemistry 1999, 50, 267. One
milligram of 4 dissolved in MeOH (2 ml) was mixed with 3% HCl (5 ml) and
stirred at room temperature. Then MeOH was evaporated and ethyl acetate
was used to extract the aglycone. The residue was analyzed by TLC and HPLC
by direct comparison with the reference sample (1). The aqueous fraction was
0
0
000
0
00
000
000
000
000
00
(
C-4 ), 63.525 (C-6 ), 44.603/44.462 (C-3), 26.901 (C-5 ), 23.488/23.425 (C-
00
00
), 18.965 (C-4 ). HR-ESI-MS [M+H] : (m/z) 503.1932 (calcd for
+
+
1
C
26
H
30
O
10 + H : 503.1912).
2 4
treated with BaCl , resulting in a white precipitate presumed to be BaSO .
1
1
5. Kim, H. J.; Kim, S.-H.; Kang, B. Y.; Lee, I.-S. Arch. Pharm. Res. 2008, 31, 1241.
6. Wu, T. S.; Hsu, M. Y.; Damu, A. G.; Kuo, P. C.; Su, C. R.; Li, C. Y.; Sun, H. D.
Heterocycles 2003, 60, 397.
Therefore, the structure of the more polar compound was identified as 8-
prenylnaringenin-7-sulfate (4).
20. Cerniglia, C. E.; Freeman, J. P.; Mitchum, R. K. Appl. Environ. Microbiol. 1982, 43,
1070.
21. Ibrahim, A.-R. S. Phytochemistry 2000, 53, 209.
22. Ibrahim, A.-R. S.; Galal, A. M.; Ahmed, S. A.; Mossa, G. S. Chem. Pharm. Bull.
2003, 51, 203.
23. Ibrahim, A.-R. S.; Abul-Hajj, Y. J. Appl. Environ. Microbiol. 1989, 55, 3140.
24. Ibrahim, A. K.; Radwan, M. M.; Ahmed, S. A.; Slade, D.; Ross, S. A. Phytochemistry
2010, 71, 1014.
25. Yagi, A.; Uemura, T.; Okamura, H.; Imoto, T.; Hashimoto, K. Phytochemistry
1994, 35, 885.
26. Haraguchi, H.; Ohmi, I.; Sakai, S.; Fakuda, A. J. Nat. Prod. 1996, 59, 443.
27. Grogan, G. J.; Holland, H. L. J. Mol. Catal. B: Enzym. 2000, 9, 1.
28. Huszcza, E.; Bartma n´ ska, A.; Klessen, S. In: Proceedings of the 9th International
Commodity Science Conference, 2007, 817.
29. Huszcza, E.; Bartma n´ ska, A.; Tronina, T. Z. Naturforsch. 2008, 63c, 557.
30. Kim, H. J.; Lee, I. S. J. Nat. Prod. 2006, 69, 1522.
31. Herath, W.; Mikell, J. L.; Hale, A. L.; Ferreira, D.; Khan, I. A. Chem. Pharm. Bull.
2006, 54, 320.
000
D
-4 -O-methylglucopyranoside (3) was isolated as
1
7. 8-Prenylnaringenin 7-O-b-
light yellow crystals (36 mg, 32.9%). UV kmax (MeOH) nm: 279, 343; 1H NMR
(
2
6
acetone-d ): d = 12.088/12.073 (1H, s, 5-OH), 7.407/7.403 (2H, d, J = 8.5 Hz, H-
0
0
0
0
,6 ), 6.904 (2H, d, J = 8.5 Hz, H-3 ,5 ), 6.269/6.266 (1H, s, H-6), 5.494/5.473
0
0
(
1H, t, J = 3.4 Hz, H-2), 5.207 (1H, br t, J = 6.9 Hz, H-2 ), 5.059/5.034 (1H, d,
000
000
J = 7.7 Hz, H-1 ), 3.852 (1H, d, J = 11.6 Hz, H-6 ), 3.705 (1H, dd, J = 11.8, 4.6 Hz,
H-6 ), 3.654 (1H, m, H-5 ), 3.563 (1H, s, 4 -OCH ), 3.559 (1H, m, 3 ), 3.547
000
000
000
000
3
000
000
00
(
1H, m, H-2 ), 3.534 (1H, m, 4 ), 3.221 (2H, m, 1 ), 3.183 (1H, m, 3ax), 2.829/
0
0
00 13
2
d
1
.801 (1H, m, 3 equiv), 1.610 (3H, s, 5 ), 1.605 (3H, s, 4 ). C NMR (acetone-
): d = 199.315/199.275 (C-4), 165.296/165.263 (C-7), 163.954/163.882 (C-5),
6
0
0
0
0
61.354/161.316 (C-9), 159.643 (C-4 ), 132.224 (C-3 ), 131.854 (C-1 ), 129.872
0
0
00
0
0
(
C-2 ,6 ), 124.783 (C-2 ), 117.154 (C-3 ,5 ), 111.296/111.246 (C-8), 105.438/
000
1
8
6
1
5
05.407 (C-10), 102.215/102.101 (C-1 ), 97.216/97.188 (C-6), 80.982 (C-2),
0
00
000
000
000
0.808/80.787 (C-4 ), 78.999 (C-3 ), 78.143 (C-5 ), 75.900 (C-2 ), 62.967 (C-
000
00
00
), 61.533 (O-CH
3
), 44.593/44.461 (C-3), 26.904 (C-5 ), 23.480/23.419 (C-1 ),
8.962 (C-4 ). HR-ESI-MS [M+H] : (m/z) 517.2097 (calcd for C27
00
+
+
H
32
O
10 + H :
17.2068).